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Specific determination of quinidine and metabolites in biological fluids by reversed-phase high-performance liquid chromatography
JownaZ of Chromatography. 228 (1982) 366-371 Biomedical AppZications Ekevier Scientific Publishing Company, Amsterdam -
CHROMBIO.
Printed in The Netherlands
1153
Note Specific determina tion of quinidine and metabolites in biological fluids by reversed-phase high-performance liquid chromatography R. LEROYER Department
of
Analytical
Chemistry,
Faculty
of
Pharmacy,
Unil;ersity
of Caen. Caen
(France)
and C. JARREAU Department
and M. PAYS’-* of Clinical Biochemktry.
Versailles Hospital Center.
7801 I Versailles (France)
(First received June 22nd. 1981; revised manuscript received October 21st. 1981)
.
As in the case of other drugs with a narrow therapeutic index, the determination 0% quinidine in biological fluids is justified by the good correlation between the zero, therapeutic or toxic effects of this ant&rhythmic drug and the blood levels. That a specific assay is needed has been shown elsewhere by the presence of pharmacologically active metabolites [ 3(S)-hydroxyquinidine, 2’-quinidone, quinidine N-aside] which may be present in high concentration in the plasma of treated subjects [l-3] , as also is dihydroquinidine, an impurity that occurs in the preparation of quinidine and which can amount to 20% of the administered dose [4]. In comparison to spectrofluorimetric assays that use protein precipitation-fluorescence (PPF) 15, 61 or extractionfluorescence (EF) [‘i-!3] techniques, chromatographic methods have the advantage of being more sensitive and specific. However, thin-layer chromatography (TLC) [10-131 and gas-liquid chromatography (GLC) [14-183 are tedious, time-consuming, and difficult. An extraction phase is necessary, and the choice of polarity of the solvent influences the co-extraction of the metabelites both qualitatively an&quantitatively. Lastly, in GLC methods, derivatisation using flash-methylatiou- is often carried out [ 14-16]_ Recent publications *Present address: Department of Clinical Biochemistry, Versailles, 78150 Le Chesnay, France. 0378-4347/82/0000-0000/$02.75
Hopital A. Mignot,
177,
Q 19S2 Elsevier Scientific Publishing Company
rue de
about quinidine assays in biological fluids or in pharmaceutical .formulations describe. high-performance liquid-&romatographid (HPLC): procedures because of the:o_bvious ~advanta&s:. rapidity; .specificify ..and sensitivity. Th&e methods indicate either the-use of a normal-ph&se~column with alkaline extraction, UV [B-22] or fiuorimetric [23] detection, or-the use of a reversed-phase column with UV 124-281 or fluorimetric [29-35] detection. In the latter, injection may be carried out either with plasma direct [29], or with a supematant obtained after protein precipitation and centrifugation [25, 33, 341, or with an aliquot of residue reconstituted in the mobile phase after alkaline extraction with an appropriate solvent and evaporation to dryness 124, 26-28, 30-32, 35]_ The HPLC method described here uses direct protein precipitation of samples with acetonitrile, a C Is reversed-phase column and fluorimetric detection. The choice of mobile phase allows internal standardization with quinine, whose fluorescence characteristics are similar to those of quinidine and dibydroquinidine. Excellent- separation of these three drugs and of polar quinidine metabolites using a rapid and easy technique with sensitive detection represents the advantage of this method. MATERIAIS
Reagents
AND METHODS
and standards
Ah solvents used are analytical ragent grade (E. Merck, Darmstadt, G.F.R.): acetonitrile for spectroscopy, methanol, acetic acid 100% Phosphate buffer (0.05 M, pH 7.4) is made up in distilled water. Pure standards of quinidine, 3(S)-hydroxyquinidine, dihydroquinidine (as bases) are obtained from Nativelle (Paris, France), and quinine was in sulfate form (Qnz - P&SO4 - 2Hz0). Standard solutions
Stock solutions of quinidine, 3(S)-hydroxyquinidine and dihydroxyquinidine were prepared in methanol at concentrations of 1 g/l (stable for 2 months at 4“C) and 100 mg/l (stable for 15 days at 4°C). Working solutions, at concentrations between 0 and 5 mg/l, were prepared by dilution of the stock solution in a mixture (l:l, v/v) of acetonitrile and aqueous .phosphate buffer (0.05 ikf, pH 7.4). Stock solution of quinine in methanol at a concentration of 1 g/l (as base) is prepared with quinine sulfate and is used to prepare a standard solution of quinine at 2-5 mg/l in acetonitrile. Chromatographic
conditiorzs
The technique is carried out on a Chromatem apparatus, equipped with an Altex 210 pump, a Rheodyne 7010 injector with a 20-~1 loop, a Waters CIs PBondapak column 30 cm x 3.9 mm (particle size 10 pm), a Schoeffel FS 970 spectrofiuorimeter (hex = 340 nm, A,, = cut-off filter of 418 nm), set at 0.5 PA full-scale sensitivity and a time-constant of 6 sec. The mobile phase was a degassed mixture of acetonitrile-acetic acidiw+er (10:4:86) with a flow-rate of 2.5 ml/mm
368
To precipitate proteins, 100 ~1 of plasma are added to 100 ~1 of a solution of quinine (25 mg/l) in acetonitrile, After closing with Parafilm, the mixture is gently mixed on a Vortex for about 1 min, and centrifuged at 1200 g for 10 min; 16 ~1 of the resultant clear supematant are injected directly on the column. RESULTS
AND
DISCUSSION
Using the conditions described, the chromatographic resolution of the different compounds is achieved in 10 mm, with the following retention times (Fig. la): 3(S)-hydroxyquinidine tR = 2-5 min, quinidine tR = 6.2 min, quinine tR = 7.7 min, dihydroquinidine tR = 8.8 min. On typical chromatograms obtained from plasma of quinidine-treated subjects (Fig. lb). a first peak (tR = l-8 mm) is found which corresponds to one (or several) unidentified metabelite(s)_ A second peak (tR = 4.8 min) coming just before the quinidine could, according to Reece and Peikert 1331 be tentatively assigned to the recently identified N-oxide [3] whose complete structure has not yet been established. In the chromatographic conditions described, we cannot detect 2’-quinidinone because of its weak fluorescence and its low plasma concentrations [30] _
0a
2
4
5
I l
0
II 2
4
L 4
l
6
8
10
12
t
0
2
4
6
8
10
12
min min Fig. I_ Cbromatograms of (a) loaded plasma at a concentration of 2 mg/l in 2.4 and 6, and (b) plasma from a treated patient receiving 4 X_ 165 mg of quinidine base every 24 h in arabogalactane sulfate form (Longacor)_ 1 and 3 = unidentified. metabolites, 2 = 3(S)hydroquinidine (O-47 mg/l), 4 = quinidine (2.58 mg/l), 5 = quinine (2.5 mg/l), 6 = dihydroquinidine.
369
However; if a-specific-extrat&iofi. 1361 of p&&a or urine{ is used, this .metaboli?%c& be deeted (TV = 16;2 min).: = ._ _--.. -. StaArd CurV& are based oti-lo&de&plasma Samples treated with acet&itrile in~hdin~ the- q&ine internal stand&d; dh&en- .for its structural relationship and its identical fluorescencd with. quinidine, and for its suitable .retention time. AS intern&standard, other tested compounds [cinchonine; cinchonidine, 3(R)hydroxy&inidine; --.dihydroquinidinone] show unsatisfactory resolution or their fluorescence is too weak. Standard curves for quinidine, dihydroqbinidine and 3(S)-hydroxyquinidine were -linear for concen@ations up to 5 mg/l, verified by measuring the peak height ratio. The calibration graphs can be expressed by the equations y = 9-172x - O-111, y = 7.054~ - 0.214, and y = 17.391x + 0.122, respe+ively. Correlation coefficients were typically. 0.996, 0.997 and 0.995, respectively. Recovery is 100 i 3%, when the concentrations of plasma loaded with different compounds are compared with standard solutions in a mixture (1:l) of acetonitrile and aqueous 0.05 M phosphate buffer. This shows that there is no loss by adsorption on the precipitate. The sensitivity for each component was 50 ng/ml_ Intra-assay reproducibility for the assay of quinine, quinidine and metabelites was determined by assaying six replicate plasma samples containing added amounts of drug and metabolites at concentrations ranging from 0.5 to 5 mg/l_ For each concentration the within-run precision was determined with a coefficient of variation of less than 3%. The day-to-day precision was determined on six consecutive days using frozen samples of plasma at levels ranging from 0.5 to 5.mg/l (Table I)_ TABLE1 VARIATIONINREPLICATESTANDARDSFROMSPIKEDPLASMA Withimmn
Day-to-day
Concentration
Concentration
0.5 QuInineS.D. C-V_<%) QuinidineS_D_ C.V.
0.06 1.2 0.07 2 0.14 1.5
1
2.5
0.09 1 0.09 1.3 0.12 0.6
0.15 1.9 0.22 1.8 0.09 1.1 0.12 0.5
5
0.32 1.3 0.2 1.2
0.5
O-15 3.3 0.13 3.9 0.36 4.1
1
2.5
5
0.31 3.5 0.23 3.4 0.7 4
0.30 1.9 1.55 6.7 0.89 5.1 2.3 5.2
2-43 3.3 1.9 5.4
The excitation wavelength (340 nm) was chosen to avoid possible interference at 240 nm by other endogenous or exogenous plasma compounds, and to obtain in a greater fluorescence intensity that at 280 nm, With a 41%nm cut-off filter, the four compounds [3(S)-hydroxyquinidine, quinidine, quinine, ~ydroquinidine] have, at the same concentration, equivalent fluorescence 130, 331, and injection of drug-free plasma shows only a small front peak. In comparison with other reversed-phase techniques, we use direct protein pr&pit&on of samples; thus risk of clogging the column with plasma injection [29] or long and non-quantitative extraction. steps are avoided [24, 26-28,
370
30-32, 353 _ As deproteinizin g agent, acetonitrile, a component of the mobile phase, is suitable [33], but when cinchonine or cinchonidine have bad fluorescence characteristics and high impurity concentrations 130,331, quinine seems to be the most convenient internal standard, Its good resolution with quinidine and dihydroquinidine is possible in a simpie isocratic system on a non-thermostated Cl8 cohmzn, when others methods show interference between these drugs [24, 25,31, 333, have only external standardization 125. 29, 321, or use dihydroquinidine, a common impurity of quinidine [34], as internal standard. Recently, enzyme immunoassay (EIA) has been compared with fluorescence spectroscopy 1373 and HPLC 138, 393, and if a good correlation exists between EIA and EF, higher quinidine concentrations are obtained with ELA comparzd with HPLC, which is explained by cross-reactions of antibody with quinidine metabolites or dihydroquinidine_ The use of a specific method for quinidine assay is necessary for pharmacokinetic studies [40, 41] as well as for drug monitoring. In a steady-state situation blood concentrations of metabolites may be high and differences between non-specific (PPF, EF) and specific methods (HPLC, TLC, GLC) may be as high as 157% [42] because of co-extraction of metabolites (of the order of 60% for 3(s)-hydroxyquinidine, and 10% for quinidine N-oxide [22, 30]_
REFERENCES F-L Carroll, D. Smith and M-E. Wall, J. Med. Chem., 17 (1974) 985. D-E- Drayer, C-E- Cook and M-M- Reideabeg, Clin_ Res_, 24 (1976) 623k T.W. Guentert, P.E. Coates and S. Riegehnan, Amer. Pharxn. Assoc., Academy PharmaceuticaiScienceMeeting,Abstracts 8-l (1978) 137. 4 U.S. Pharznacopeia, Mack Publishing Co., Easton, PA, 19th ed., 1975,434. B.B. Brodie and S: Udenfriend, J- Pharmacol. Exp_ Ther., 78 (1943) 154. s” P.H. Balatre, C. Lefevre and C-F. Merlen, Ann_ Biol_ Clin., 18 (1960) 228. 7 A-L- Edgar and M. Sokolow, J- Lab. Clin. Med., 36 (1950) 478. 8 G. Cramer and B. bsakson, Stand. J. Clin. Invest., 15 (1963) 553. 9 J_ Armand and A Badinand, Ann. Biol. Clin., 30 (1972) 599. 10 G. KiZrtel and A_ Korhonen, J. Chromatogr-, 37 (1968) 70. 11 G_ HZ&e1 and A. Harjanne, Clin. Chim_ Acta, 23 (1969) 289. 12 B. Wesley-Hadzija and A-M_ Mattocks, J. Chromatogr-, 144 (1977) 223. 13 CT_ Ueda, D.S. Hirschfeld, M_M_ Scheinman, M. Rowland, B-J. Williamson and B-S. Dzindzio, Clin. Pharmacol. Ther-, 19 (1976) 30. 14 K.K. Midha and C. Charette, J_ Pharm- Sci., 63 (1974) 1244. 15 J.L. Valentine, P_ Driioll, E-L. Hamburg and E.D. Thompson, J. Pharm- Sci., 65 (1976) 9616 D.H. Huffman and C.E. Ignite, Clin- Chem., 22 (1976) 810. 17 IM_A_Moulin and H_ Kinsun, Clin. Chim. -4cta, 75 (1977) 491. 18 J.G. Leferinck, R.A.A. Maes, I. Sunshine and R.B. Forney, J. Anal. Toxicol., l(l977) 62. 19 M.A. Peat and T.A. Jennison, Clin. Chem., 24 (1978) 2166. 20 R-G. Achari, J.L. Baldridge, T.R. Koziol and L. Yu, J_ Chromatogr., Sci., 16 (1978) 271. 21 R-E- Kates, D-W- MacKennon 2nd T-J_ Cornstock, J_ Pharm_ ScL, 67 (1978) 269. 22 T.W. Guentert, P.E. Coates, R-A. Upton, D-L_ Combs and S. Riegehnan, J. Chromatogr., 162 (1979) 59. 23 S- Sved, LJ- MacGilveray and N- Beaudoin, J. Chromatogr., 145 (1978) 437. 1 2 3
_-_ _~ 24. 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42
371: W.G..&out&&l, B. K&&ski and P.K. Narang, &I_ &hen+, 23 (1977) 2030. J&. P&w&s and W. Ssdee, Clin. &em.; 24 (1978) 289, M.R. Boriora, T-W_ Guen%ert, R.A. Upton and S...Rie&lmah._ &in. C&n. Acta, 91 : (1979).27?_ J-G. Flood. G.N. l&e& &d R.B. M&&&, Clin. &h&m., 28 (198Oj iSi_ S.E. Barrow,. A.A. Taylor, E.C. Homing and M.G. Homing, J. Chromatogr., 181(1980) 219. K_A Conrad, B-L. Molk and CA Chidsey, Circulation, 55 (1977) 1. D-E_ Drayer, KM_ Restivo and M.M. Reidenberg, J. Lab. Clin. Med., 90 (1977) 816. N. Weidner, J.H. Landenson, L. Larson, G. Kessler and J.M. MacDonald, Clin. Chim. Acta, 91(1979) 7. B-J. Kline, V-A Turner and W-H_ Barr, Anal. Chem., 51(1979) 449. P-A. Reece and M. Peikert, J. Chkomatdgr+ 181(1980) 207. T-W. Guentert and S. Qe, J. PhFacoL Exp. Ther., 215 (1980) 165. T-W. Guentert, A Rakhit, R.A Upton and S..Riegelman, J. Chromatogr., 183 (1980) 514. D.E. Drayer, D.T. Lowe&ha& K.M. Restive, A. Schwartz, C.E. Cook and M.M. Riedenberg, Clin. Pharmacol. Ther., 24 (1978) 31. K.K_ Batra, J. Omand and R.C. Baselt, Clin. Chem., 27 (1981) 780. H-R. Ha, G. Kewitz, F. Wewk and F. Follath, J_ Clin. Pharmacol-, ll(l981) 312. D-E_ Drayer, B. Lorenzo and M.F. Reidenberg, Clin. Chem., 27 (1981) 308. T.W. Gutinter& R.A. Upton, N.G. Holford and S. Riegelman, J. Pharmacokin. Biopharm., 7 (1979) 303. T.W. Guentert, N-G. Holford, P.E. Co&es, R.A. Upton and S. Riegelman, J. Pharmacokin. Biopharm;, ‘7 (1979) 315. T-W. Guentert and S. Riegelman, Clin. Chem., 24 (1978) 2065.
CHROMBIO.
Printed in The Netherlands
1153
Note Specific determina tion of quinidine and metabolites in biological fluids by reversed-phase high-performance liquid chromatography R. LEROYER Department
of
Analytical
Chemistry,
Faculty
of
Pharmacy,
Unil;ersity
of Caen. Caen
(France)
and C. JARREAU Department
and M. PAYS’-* of Clinical Biochemktry.
Versailles Hospital Center.
7801 I Versailles (France)
(First received June 22nd. 1981; revised manuscript received October 21st. 1981)
.
As in the case of other drugs with a narrow therapeutic index, the determination 0% quinidine in biological fluids is justified by the good correlation between the zero, therapeutic or toxic effects of this ant&rhythmic drug and the blood levels. That a specific assay is needed has been shown elsewhere by the presence of pharmacologically active metabolites [ 3(S)-hydroxyquinidine, 2’-quinidone, quinidine N-aside] which may be present in high concentration in the plasma of treated subjects [l-3] , as also is dihydroquinidine, an impurity that occurs in the preparation of quinidine and which can amount to 20% of the administered dose [4]. In comparison to spectrofluorimetric assays that use protein precipitation-fluorescence (PPF) 15, 61 or extractionfluorescence (EF) [‘i-!3] techniques, chromatographic methods have the advantage of being more sensitive and specific. However, thin-layer chromatography (TLC) [10-131 and gas-liquid chromatography (GLC) [14-183 are tedious, time-consuming, and difficult. An extraction phase is necessary, and the choice of polarity of the solvent influences the co-extraction of the metabelites both qualitatively an&quantitatively. Lastly, in GLC methods, derivatisation using flash-methylatiou- is often carried out [ 14-16]_ Recent publications *Present address: Department of Clinical Biochemistry, Versailles, 78150 Le Chesnay, France. 0378-4347/82/0000-0000/$02.75
Hopital A. Mignot,
177,
Q 19S2 Elsevier Scientific Publishing Company
rue de
about quinidine assays in biological fluids or in pharmaceutical .formulations describe. high-performance liquid-&romatographid (HPLC): procedures because of the:o_bvious ~advanta&s:. rapidity; .specificify ..and sensitivity. Th&e methods indicate either the-use of a normal-ph&se~column with alkaline extraction, UV [B-22] or fiuorimetric [23] detection, or-the use of a reversed-phase column with UV 124-281 or fluorimetric [29-35] detection. In the latter, injection may be carried out either with plasma direct [29], or with a supematant obtained after protein precipitation and centrifugation [25, 33, 341, or with an aliquot of residue reconstituted in the mobile phase after alkaline extraction with an appropriate solvent and evaporation to dryness 124, 26-28, 30-32, 35]_ The HPLC method described here uses direct protein precipitation of samples with acetonitrile, a C Is reversed-phase column and fluorimetric detection. The choice of mobile phase allows internal standardization with quinine, whose fluorescence characteristics are similar to those of quinidine and dibydroquinidine. Excellent- separation of these three drugs and of polar quinidine metabolites using a rapid and easy technique with sensitive detection represents the advantage of this method. MATERIAIS
Reagents
AND METHODS
and standards
Ah solvents used are analytical ragent grade (E. Merck, Darmstadt, G.F.R.): acetonitrile for spectroscopy, methanol, acetic acid 100% Phosphate buffer (0.05 M, pH 7.4) is made up in distilled water. Pure standards of quinidine, 3(S)-hydroxyquinidine, dihydroquinidine (as bases) are obtained from Nativelle (Paris, France), and quinine was in sulfate form (Qnz - P&SO4 - 2Hz0). Standard solutions
Stock solutions of quinidine, 3(S)-hydroxyquinidine and dihydroxyquinidine were prepared in methanol at concentrations of 1 g/l (stable for 2 months at 4“C) and 100 mg/l (stable for 15 days at 4°C). Working solutions, at concentrations between 0 and 5 mg/l, were prepared by dilution of the stock solution in a mixture (l:l, v/v) of acetonitrile and aqueous .phosphate buffer (0.05 ikf, pH 7.4). Stock solution of quinine in methanol at a concentration of 1 g/l (as base) is prepared with quinine sulfate and is used to prepare a standard solution of quinine at 2-5 mg/l in acetonitrile. Chromatographic
conditiorzs
The technique is carried out on a Chromatem apparatus, equipped with an Altex 210 pump, a Rheodyne 7010 injector with a 20-~1 loop, a Waters CIs PBondapak column 30 cm x 3.9 mm (particle size 10 pm), a Schoeffel FS 970 spectrofiuorimeter (hex = 340 nm, A,, = cut-off filter of 418 nm), set at 0.5 PA full-scale sensitivity and a time-constant of 6 sec. The mobile phase was a degassed mixture of acetonitrile-acetic acidiw+er (10:4:86) with a flow-rate of 2.5 ml/mm
368
To precipitate proteins, 100 ~1 of plasma are added to 100 ~1 of a solution of quinine (25 mg/l) in acetonitrile, After closing with Parafilm, the mixture is gently mixed on a Vortex for about 1 min, and centrifuged at 1200 g for 10 min; 16 ~1 of the resultant clear supematant are injected directly on the column. RESULTS
AND
DISCUSSION
Using the conditions described, the chromatographic resolution of the different compounds is achieved in 10 mm, with the following retention times (Fig. la): 3(S)-hydroxyquinidine tR = 2-5 min, quinidine tR = 6.2 min, quinine tR = 7.7 min, dihydroquinidine tR = 8.8 min. On typical chromatograms obtained from plasma of quinidine-treated subjects (Fig. lb). a first peak (tR = l-8 mm) is found which corresponds to one (or several) unidentified metabelite(s)_ A second peak (tR = 4.8 min) coming just before the quinidine could, according to Reece and Peikert 1331 be tentatively assigned to the recently identified N-oxide [3] whose complete structure has not yet been established. In the chromatographic conditions described, we cannot detect 2’-quinidinone because of its weak fluorescence and its low plasma concentrations [30] _
0a
2
4
5
I l
0
II 2
4
L 4
l
6
8
10
12
t
0
2
4
6
8
10
12
min min Fig. I_ Cbromatograms of (a) loaded plasma at a concentration of 2 mg/l in 2.4 and 6, and (b) plasma from a treated patient receiving 4 X_ 165 mg of quinidine base every 24 h in arabogalactane sulfate form (Longacor)_ 1 and 3 = unidentified. metabolites, 2 = 3(S)hydroquinidine (O-47 mg/l), 4 = quinidine (2.58 mg/l), 5 = quinine (2.5 mg/l), 6 = dihydroquinidine.
369
However; if a-specific-extrat&iofi. 1361 of p&&a or urine{ is used, this .metaboli?%c& be deeted (TV = 16;2 min).: = ._ _--.. -. StaArd CurV& are based oti-lo&de&plasma Samples treated with acet&itrile in~hdin~ the- q&ine internal stand&d; dh&en- .for its structural relationship and its identical fluorescencd with. quinidine, and for its suitable .retention time. AS intern&standard, other tested compounds [cinchonine; cinchonidine, 3(R)hydroxy&inidine; --.dihydroquinidinone] show unsatisfactory resolution or their fluorescence is too weak. Standard curves for quinidine, dihydroqbinidine and 3(S)-hydroxyquinidine were -linear for concen@ations up to 5 mg/l, verified by measuring the peak height ratio. The calibration graphs can be expressed by the equations y = 9-172x - O-111, y = 7.054~ - 0.214, and y = 17.391x + 0.122, respe+ively. Correlation coefficients were typically. 0.996, 0.997 and 0.995, respectively. Recovery is 100 i 3%, when the concentrations of plasma loaded with different compounds are compared with standard solutions in a mixture (1:l) of acetonitrile and aqueous 0.05 M phosphate buffer. This shows that there is no loss by adsorption on the precipitate. The sensitivity for each component was 50 ng/ml_ Intra-assay reproducibility for the assay of quinine, quinidine and metabelites was determined by assaying six replicate plasma samples containing added amounts of drug and metabolites at concentrations ranging from 0.5 to 5 mg/l_ For each concentration the within-run precision was determined with a coefficient of variation of less than 3%. The day-to-day precision was determined on six consecutive days using frozen samples of plasma at levels ranging from 0.5 to 5.mg/l (Table I)_ TABLE1 VARIATIONINREPLICATESTANDARDSFROMSPIKEDPLASMA Withimmn
Day-to-day
Concentration
Concentration
0.5 QuInineS.D. C-V_<%) QuinidineS_D_ C.V.
0.06 1.2 0.07 2 0.14 1.5
1
2.5
0.09 1 0.09 1.3 0.12 0.6
0.15 1.9 0.22 1.8 0.09 1.1 0.12 0.5
5
0.32 1.3 0.2 1.2
0.5
O-15 3.3 0.13 3.9 0.36 4.1
1
2.5
5
0.31 3.5 0.23 3.4 0.7 4
0.30 1.9 1.55 6.7 0.89 5.1 2.3 5.2
2-43 3.3 1.9 5.4
The excitation wavelength (340 nm) was chosen to avoid possible interference at 240 nm by other endogenous or exogenous plasma compounds, and to obtain in a greater fluorescence intensity that at 280 nm, With a 41%nm cut-off filter, the four compounds [3(S)-hydroxyquinidine, quinidine, quinine, ~ydroquinidine] have, at the same concentration, equivalent fluorescence 130, 331, and injection of drug-free plasma shows only a small front peak. In comparison with other reversed-phase techniques, we use direct protein pr&pit&on of samples; thus risk of clogging the column with plasma injection [29] or long and non-quantitative extraction. steps are avoided [24, 26-28,
370
30-32, 353 _ As deproteinizin g agent, acetonitrile, a component of the mobile phase, is suitable [33], but when cinchonine or cinchonidine have bad fluorescence characteristics and high impurity concentrations 130,331, quinine seems to be the most convenient internal standard, Its good resolution with quinidine and dihydroquinidine is possible in a simpie isocratic system on a non-thermostated Cl8 cohmzn, when others methods show interference between these drugs [24, 25,31, 333, have only external standardization 125. 29, 321, or use dihydroquinidine, a common impurity of quinidine [34], as internal standard. Recently, enzyme immunoassay (EIA) has been compared with fluorescence spectroscopy 1373 and HPLC 138, 393, and if a good correlation exists between EIA and EF, higher quinidine concentrations are obtained with ELA comparzd with HPLC, which is explained by cross-reactions of antibody with quinidine metabolites or dihydroquinidine_ The use of a specific method for quinidine assay is necessary for pharmacokinetic studies [40, 41] as well as for drug monitoring. In a steady-state situation blood concentrations of metabolites may be high and differences between non-specific (PPF, EF) and specific methods (HPLC, TLC, GLC) may be as high as 157% [42] because of co-extraction of metabolites (of the order of 60% for 3(s)-hydroxyquinidine, and 10% for quinidine N-oxide [22, 30]_
REFERENCES F-L Carroll, D. Smith and M-E. Wall, J. Med. Chem., 17 (1974) 985. D-E- Drayer, C-E- Cook and M-M- Reideabeg, Clin_ Res_, 24 (1976) 623k T.W. Guentert, P.E. Coates and S. Riegehnan, Amer. Pharxn. Assoc., Academy PharmaceuticaiScienceMeeting,Abstracts 8-l (1978) 137. 4 U.S. Pharznacopeia, Mack Publishing Co., Easton, PA, 19th ed., 1975,434. B.B. Brodie and S: Udenfriend, J- Pharmacol. Exp_ Ther., 78 (1943) 154. s” P.H. Balatre, C. Lefevre and C-F. Merlen, Ann_ Biol_ Clin., 18 (1960) 228. 7 A-L- Edgar and M. Sokolow, J- Lab. Clin. Med., 36 (1950) 478. 8 G. Cramer and B. bsakson, Stand. J. Clin. Invest., 15 (1963) 553. 9 J_ Armand and A Badinand, Ann. Biol. Clin., 30 (1972) 599. 10 G. KiZrtel and A_ Korhonen, J. Chromatogr-, 37 (1968) 70. 11 G_ HZ&e1 and A. Harjanne, Clin. Chim_ Acta, 23 (1969) 289. 12 B. Wesley-Hadzija and A-M_ Mattocks, J. Chromatogr-, 144 (1977) 223. 13 CT_ Ueda, D.S. Hirschfeld, M_M_ Scheinman, M. Rowland, B-J. Williamson and B-S. Dzindzio, Clin. Pharmacol. Ther-, 19 (1976) 30. 14 K.K. Midha and C. Charette, J_ Pharm- Sci., 63 (1974) 1244. 15 J.L. Valentine, P_ Driioll, E-L. Hamburg and E.D. Thompson, J. Pharm- Sci., 65 (1976) 9616 D.H. Huffman and C.E. Ignite, Clin- Chem., 22 (1976) 810. 17 IM_A_Moulin and H_ Kinsun, Clin. Chim. -4cta, 75 (1977) 491. 18 J.G. Leferinck, R.A.A. Maes, I. Sunshine and R.B. Forney, J. Anal. Toxicol., l(l977) 62. 19 M.A. Peat and T.A. Jennison, Clin. Chem., 24 (1978) 2166. 20 R-G. Achari, J.L. Baldridge, T.R. Koziol and L. Yu, J_ Chromatogr., Sci., 16 (1978) 271. 21 R-E- Kates, D-W- MacKennon 2nd T-J_ Cornstock, J_ Pharm_ ScL, 67 (1978) 269. 22 T.W. Guentert, P.E. Coates, R-A. Upton, D-L_ Combs and S. Riegehnan, J. Chromatogr., 162 (1979) 59. 23 S- Sved, LJ- MacGilveray and N- Beaudoin, J. Chromatogr., 145 (1978) 437. 1 2 3
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